The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, gram-positive rods. The genus comprises several species which include, but are not limited to, Mycobacterium africanum, M. avium, M. bows, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. microti, M. scrofidaceum, M. paratuberculosis and M. tuberculosis. Certain of these organisms are the causative agents of disease. For the first time since 1953, cases of mycobacterial infections are increasing in the United States. Of particular concern is tuberculosis, the etiological agent of which is M. tuberculosis. However, infections caused by Mycobacteria other than tuberculosis (MOTT) have also been increasing. Many of these new cases are related to the AIDS epidemic, which provides an immune compromised population which is particularly susceptible to infection by Mycobacteria. Mycobacterium avium, Mycobactenum kansasii and other non-tuberculosis mycobacteria have been found as opportunistic pathogens in HIV-infected and other immune compromised patients. There is an increasing need for rapid diagnosis of these infections, as they can disseminate rapidly and may be fatal within a short period of time.
At the present time the diagnosis of mycobacterial infections is dependent on acid-fast staining and cultivation of the organism, followed by biochemical assays. These procedures are time-consuming, and a typical diagnosis using conventional culture methods can take as long as six weeks. Automated culturing systems such as the BACTEC system (Becton Dickinson Microbiology Systems, Sparks, Md.) can decrease the time for diagnosis to one to two weeks. However, there is still a need to reduce the time required for diagnosing mycobacterial infections to less than a week, preferably to about one day. Oligonucleotide probe based assays such as Southern hybridizations or dot blots are capable of returning a rapid result (i.e., in one day or less). Genus- and species-specific primers may also be used in direct assays of clinical samples by nucleic acid amplification. However, both of these rapid detection and identification methods require oligonucleotide probes or primers which are specific for the genus Mycobacteria (tuberculosis and non-tuberculosis) or specific for a particular mycobacterial species if specific identification of the organism is desired.
Conventional laboratory identification of Mycobacterium kansasii relies upon biochemical testing and determination of growth characteristics. These include catalase production, urease activity, TWEEN hydrolysis, nitrate reduction and the ability of the bacterium to produce pigment when exposed to light (photochromogenicity). Because several other mycobacterial species exhibit a similar biochemical profile, photochromogenicity is customarily relied upon as the conclusive characteristic for identification of Mycobacterium kansasii. However, determination of photochromogenicity requires a pure culture of the organism and this phenotype can be variable, subjective and difficult to determine reliably. For these reasons, there have been attempts to identify Mycobacterium kansasii by species-specific hybridization or nucleic acid amplification using oligonucleotide probes. Z. H. Huang, et al. (1991. J. Clin. Microbiol. 29, 2125-2129) have reported a DNA probe obtained from a genomic library with a degree of species-specificity for Mycobacterium kansasii. This clone (pMK1-9) showed some cross-hybridization with other species, including M. gastri, and did not detect a genetically distinct subgroup of M. kansasii. The nucleotide sequence of pMK1-9 was not reported, nor was the gene from which it may have been derived identified. B. C. Ross, et al. (1992. J. Clin. Microbid. 30, 2930-2933) also reported identification of genetically distinct subspecies of M. kansasii using the pMK1-9 probe, a 65kDa antigen gene probe and a commercial DNA probe test employing probes which specifically hybridize to rRNA (ACCU-PROBE, Gen-Probe, San Diego, Ca.). Amplification primers which hybridize to the 16S rRNA gene were used to sequence and compare the 16S rRNA gene sequences of the M. kansasii variants studied. M. Yang, et al. (1993. J. Clin. Microbid. 31, 2769-2772) have reported isolation of a sequence from a clinical isolate which, when used as a hybridization probe, exhibits M. kansasii species-specificity. This probe (p6123) hybridized to all M. kansasii strains tested, including the subgroup which is pMK1-9 negative.
T. Rogall, et al. (1990. J. Gen. Microbiol. 136, 1915-1920) used the 16S rRNA sequence in a polymerase chain reaction (PCR) based sequencing strategy for identification of mycobacterial species. However, these primers could not be used to differentiate M. gastri from M. kansasii because the 16S rRNA sequence from these two species is identical in spite of their differing phenotypic characteristics. Similar studies have been published by B. Boddinghaus, et al. (1990. J. Clin. Microbiol. 28, 1751-1759), who reported oligonucleotides derived from 16S rRNA sequences which are specific for the M. tuberculosis group, i.e., M. avium-M. paratuberculosis, and M. intracellulare.
The present invention provides nucleic acid sequences useful as amplification primers for species-specific detection and identification of Mycobacterium kansasii. Species-specificity means that the inventive primers amplify a target sequence in M. kansasii nucleic acids with little or no detectable amplification of target sequences of other species of mycobacteria or of closely related microorganisms such as M. gastri, Rhodococcus rhodochrous and Nocardia asteroides. The amplification primers of the invention are derived from the p6123 sequence previously reported by Yang, et al. (1993), however, these authors had previously used only the full-length p6123 sequence to demonstrate species-specificity by probe hybridization (e.g., Southern blots). It was not known prior to the present invention whether short amplification primers could be designed which would retain species-specificity and also function as primers for SDA.
The primers may be used after culturing a sample as a means for confirming identity of the culture. Alternatively, they may be used prior to culturing for detection and identification of mycobacterial DNA using nucleic acid amplification techniques as is known in the art. In either case, the inventive primers and diagnostic methods provide a means for rapidly discriminating between M. kansasii and other species of mycobacteria, allowing the practitioner to rapidly identify this microorganism without resorting to the time-consuming phenotypic and biochemical procedures customarily relied upon. Such rapid identification of the specific etiological agent involved in a mycobacterial infection provides information which can be used to determine appropriate therapy within a short period of time.